I'm using px330 plasmid, cutting it with BbsI, and using annealed oligos that are 20 bp in length. I've used RE with cutsmart buffer. Purified it used a qiagen gel purification column.
For the annealed oligos, I've ran a RE digest to make sure I get the right cut sites, and when purifying using the qiagen purification column, I end up with less than 5ng/uL concetration. Before RE digeting, I was at a 2000ng/uL
My RE-digest mix is 40uL total, with 0.5 uL enzyme and 2uL of oligos.
Not sure if just head inactivating the digested oligos will help, or that digesting isn't necessary.
Any help is much appreciated
In theory, you can generate an oligo duplex and generate the proper compatible ends by restriction cutting, but it is a rather awkward strategy. Given the high molarity of the duplex due to its small size, unless you digest a minute quantity, you will have to use a huge amount of restriction enzyme to achieve complete digestion. Why dont you design your primers such that after annealing, they include the 4-base 5' extensions complementary to the 4-base 5' extensions left in your vector after linearization with BbsI ? Given the low cost of 20-mer oligos, it may be worth while ordering another pair of oligos for this purpose.
The way to do it is as Pierre suggested above. Keep in mind columns are not good in purifying very short fragments and you will end up losing most of your product. I would also strongly suggest not to use restriction enzymes as you will end up with inserts that can anneal to each other hence you will end up with a concatemer of your insert given its small size. Non-phosphorylated hybridized oligos will not do this since they are not phosphorylated and hence cannot ligate to each other.
while ordering your oligos, did you design your oligos with two partially complementary oligos with 4nt overhangs compatible for cloning vector or not? if you did, there is no need to cut your oligos with RE since it has the sticky ends compatible with the overhung end at your vector. if you didn't I recommend you to order your oligo again with 4nt overhangs since the BbSI is hard to work with.
Pierre Béguin thank you for your response. The primers should have a 4-base 5' and 4base 3' extension already there, but it doesn't seem to be ligating inside the vector. I got colonies, and performed a colony pcr- our primer seem to give us unspecific bands - none of which is what we are looking for. Our screening primer extends past our 25-bp insert into the vector, 3' end of the primer sticks to the vector with the following sequence: gtt - would this be causing unspecific bindings? (have already blasted the primers on the px330 plasmid)
thanks Hanna Alalam and Niloofar Khairkhah
- Badges
- Science method